Investigating the role of chromatin modifications in CRISPR/Cas9 gene editing
Date
27/07/2020Author
Kallimasioti Pazi, Eirini Margarita
Metadata
Abstract
Precisely positioned nucleosomes and heterochromatin have been
shown to impede CRISPR/Cas9 editing efficiency. Conversely, Cas9 can open
previously inaccessible regions of DNA, and transcriptionally silent targets can
usually be edited without difficulty. In order to address this paradox, we have
developed a method that exploits the allele-specific chromatin status of
imprinted genes to characterise the impact of chromatin modifications on
targeted mutagenesis. Cas9 was targeted to imprinted CpG islands in F1
hybrid mouse embryonic stem cells, and then allele-specific mutation patterns
were characterised following high throughput amplicon sequencing. Using this
novel system, we discovered that heterochromatin can impede mutagenesis
with CRISPR/Cas9, but to a degree that depends on other key experimental
parameters. Mutagenesis was impeded when Cas9 exposure was brief and
when intracellular expression of Cas9 was low, but the consequences of
chromatin modifications were minimal following prolonged exposure. The
presence of mismatches between single guide RNA and genomic target
sequence disproportionately reduced mutagenesis within heterochromatin for
some specific combinations of mismatches. This suggests that Cas9
proofreading activity, and hence off-target mutagenesis, may be impacted by
chromatin state and merits further investigation. Lastly, no effects of chromatin
modifications on the outcome of DNA repair were detected, with similar
efficiencies of homology-directed repair (HDR) and non-homologous end
joining (NHEJ) on maternal and paternal alleles. Upon further characterisation
we discovered that the majority of types of insertions and deletions generated
by NHEJ shared similar frequencies between the two alleles. Combined, my
data show that heterochromatin imposes a permeable barrier that influences
the kinetics, but not the endpoint, of CRISPR/Cas9 genome editing and
suggest that therapeutic applications involving low-level Cas9 exposure will be
particularly affected by chromatin status.